摘要
目的探讨左归降糖舒心方含药血浆对氧化低密度脂蛋白(ox-LDL)诱导巨噬细胞泡沫化和凋亡的影响及其作用机制。方法将巨噬细胞J774A.1随机分为正常组、模型组、牛磺熊去氧胆酸(TUDCA)组、左归降糖舒心方含药血浆组、西药联合含药血浆组,每组5个复孔。除正常组外其余各组以ox-LDL(100 mg/L)处理构建巨噬细胞泡沫化模型。正常组和模型组正常培养,其余各组分别用10%相应含药血浆培养24 h。CCK-8法比较各组J774A.1细胞增殖情况,计算细胞抑制率;油红O染色检测细胞内脂质蓄积水平;Western blot检测双链RNA样内质网激酶(PEPK)、活化转录因子4(ATF4)、真核翻译起始因子2α(eIF2α)、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)及半胱氨酸蛋白酶蛋白-12(Caspase-12)的表达水平。结果与正常组比较,模型组细胞抑制率增高,在ox-LDL浓度为100 mg/L时细胞抑制率为(50.06±0.09)%,有明显的脂质颗粒蓄积(P<0.01)。与模型组比较,各药物干预组细胞抑制率降低,可有效阻断ox-LDL的摄入及脂质堆积,其中左归降糖舒心方含药血浆组优于西药联合含药血浆组,与TUDCA组作用效应相当。ox-LDL可诱导细胞内质网应激相关蛋白p-PERK、p-eIF2α、ATF4、CHOP与Caspase-12表达上调。左归降糖舒心方含药血浆可显著抑制100 mg/L ox-LDL所致的p-PERK、p-eIF2α、ATF4的上调,降低内质网相关凋亡蛋白CHOP与Caspase-12的表达(P<0.01),和TUDCA组作用相当(P>0.05),其效果较西药联合含药血浆组明显(P<0.01)。结论左归降糖舒心方含药血浆能有效改善ox-LDL诱导的巨噬细胞泡沫化、细胞凋亡及内质网应激状态,其作用机制可能与调控内质网应激信号通路PERK/eIF2α/ATF4通路,降低细胞内CHOP与Caspase-12表达有关。
OBJECTIVE To investigate the effect of Zuogui Jiangtang Shuxin Formula medicated plasma on ox-LDL-induced PERK/eIF2α/ATF4 integrated stress response and activation of apoptosis key signaling molecules CHOP and Caspase-12.METHEOD J774A.1 cells were randomly divided into 5 groups:normal group,model group,TUDCA group,Zuogui Jiangtang Shuxin Formula medicated plasma group and western medicine combination(metformin+atorvastatin calcium)medicated plasma group.All the drug intervention groups were pretreated with ox-LDL to establish an experimental model for macrophage foam cell formation.The cells of drug intervention groups were cultured in 10%different drug containing plasma,respectively.Cells of normal and model groups were cultured in normal culture medium.The growth inhibition of J774A.1 were tested by CCK-8 essay after 24 h culture.Oil Red O Dyeing experiment was used to show the cellular lipid droplets.The expression of p-PERK,p-eIF2α,ATF4,CHOP and Caspase-12 were detected by Western blot.RESULTS 100 mg/L ox-LDL suppressed the growth of J774A.1 cells with an inhibitory rate of(50.06±0.09)%.Oil Red O staining showed that the model group cells had more lipid droplets than normal group(P<0.01).Drug intervention groups effectively prevented the pervasion of ox-LDL to reduce lipid accumulation.Oil red O staining showed that lipid aggregated in Zuogui Jiangtang Shuxin Formula medicated plasma,compared with western medicine combination medicated plasma group(P<0.01),and the effect was equivalent to TUDCA group(P>0.05).ox-LDL up-regulated the expression of p-PERK,p-eIF2α,ATF4,CHOP and Caspase-12.Zuogui Jiangtang Shuxin Formula medicated plasma significantly restrained the expression of p-PERK,p-eIF2αand ATF4,and apparently decreased endoplasmic reticulum associated apoptosis protein CHOP and Caspase-12 expression(P<0.01).There was no significant difference in Zuogui Jiangtang Shuxin Formula medicated plasma group and TUDCA group.And Zuogui Jiangtang Shuxin Formula medicated plasma group was superior to the western medicine combination medicated plasma group(P<0.01).CONCLUSION Zuogui Jiangtang Shuxin Formula medicated plasma can suppress foam cell formation and apoptosis in J774A.1 cells,which may be related to the regulation of PERK/eIF2α/ATF4 signaling pathway and the expression of CHOP and Caspase-12.
作者
杨金伟
赵灿
刘秀
吴勇军
喻嵘
YANG Jin-wei;ZHAO Can;LIU Xiu;WU Yong-jun;YU Rong(The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, 410208, China;Graduate School, Hunan University of Chinese Medicine, Changsha, 410208, China;Key Laboratory of Colleges and Universities, Traditional Chinese Medicine Prescription and Transformation of Medical Laboratory, Changsha, 410208, China;School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, China)
出处
《南京中医药大学学报》
CAS
CSCD
北大核心
2020年第3期370-375,共6页
Journal of Nanjing University of Traditional Chinese Medicine
基金
国家自然科学基金(81573956)
国家重点研发计划中医药现代化研究重点专项(2018YFC1704300)
湖南省研究生科研创新项目(CX2018B471)
湖南中医药大学中医学一级学科开放性基金(2018ZYX44)。
作者简介
第一作者:杨金伟,男,博士研究生,E-mail:ykingjay@stu.hnucm.edu.cn;通讯作者:喻嵘,女,教授,博士生导师,主要从事中医药防治内分泌疾病的研究,E-mail:yuron@21cn.com。
